|Número de publicación||WO1982000660 A1|
|Tipo de publicación||Solicitud|
|Número de solicitud||PCT/US1981/001098|
|Fecha de publicación||4 Mar 1982|
|Fecha de presentación||14 Ago 1981|
|Fecha de prioridad||20 Ago 1980|
|También publicado como||EP0058689A1, EP0058689A4, EP0058689B1|
|Número de publicación||PCT/1981/1098, PCT/US/1981/001098, PCT/US/1981/01098, PCT/US/81/001098, PCT/US/81/01098, PCT/US1981/001098, PCT/US1981/01098, PCT/US1981001098, PCT/US198101098, PCT/US81/001098, PCT/US81/01098, PCT/US81001098, PCT/US8101098, WO 1982/000660 A1, WO 1982000660 A1, WO 1982000660A1, WO 8200660 A1, WO 8200660A1, WO-A1-1982000660, WO-A1-8200660, WO1982/000660A1, WO1982000660 A1, WO1982000660A1, WO8200660 A1, WO8200660A1|
|Inventores||Glass Works Corning|
|Solicitante||Mosbach K, Nilsson K|
|Exportar cita||BiBTeX, EndNote, RefMan|
|Citas de patentes (14), Otras citas (8), Citada por (38), Clasificaciones (19), Eventos legales (6)|
|Enlaces externos: Patentscope, Espacenet|
The present invention relates to microcarriers. More particularly, the present invention relates to improved microcarriers and their use in the immobilization and culturing of anchorage-dependent animal cells, and to the ready removal of such cells from such micro- carriers. The surface-area limitations associated with culturing anchorage-dependent animal cells in, for example, roller bottles to a large degree have been overcome through the use of microcarriers Where the cells per se comprise the desired product, however, problems still remain relative to the efficient harvesting of such ceils.
In the past, there has been considerable interest in the immobilization of cells, particularly those of microbial origin. K. Mosbach, Ed., "Methods in Enzym ology, " Vol. 44, Academic Press, New York, 1976; and
I. Chibata and T. Tosa in L. B. Wingard et al., "Applied Biochemistry and Bioengineering, " Vol. 1, Academic Press, New York, 1976, pp. 329-357. More recently, such interest has been extended by the reported immobilization of living plant cells in suspension. P. Brodelius et al., FEBS Letters, 103, 93-97 (1979).
The use of microcarriers for culturing mammalian anchorage-dependent cells in suspension has been given increasing attention in recent years. For example, a DEAE-Sephadex microcarrier was used in some early experiments as a support for the cultivation of fibroblast-like cells derived from embryonic rabbit skin (H ceils) and diploid human embryonic lung cells (HRL cells). A. L. van Wezel, Nature, 216, 64-65 (1967). van Wezel has continued to study the use of DEAE- Sephadex microcarriers for the homogeneous cultivation of primary cells and diploid cell strains. See, e.g., A. L. van Wezel, "Microcarrier Cultures of Animal Cells," in P. F. Kruse, Jr. and M. K. Patterson, Jr., Editors, "Tissue Culture: Methods and Applications, " Academic Press, New York, 1973, pp. 372-377; A- L. van Wezel et al., Develop, biol. Standard., 42, 65 (1979), presented at the 2nd General Meeting of ESACT, Paris 1973; and A. L. van Wezel et al., Process Biochemistry, 13, 6 (1978). Interestingly, other workers also concentrated on the use of DEAE-Sephadex microcarriers. For example, Levine et al. proposed adding to the culture medium containing DEAE-Sephadex microcarrier beads a negatively charged non-nutritive component, e.g., carboxymethyl- cellulose, to compete with the positively charged sites on the microcarrier. They also screened various support (microcarrier) materials which appeared to include silica gel and several ion-exchange resins, as well as Sephadex per se. The DEAE-Sephadex beads were in the 90-105 μ range. D. W. Levine et al., "Optimizing Parameters for Growth of Anchorage-Dependent Mammalian Ceils in Microcarrier Culture," in "Cell Culture and Its Applications," Academic Press, New York, 1977, pp. 191-216. See also U.S. Patent No. 4,036,693 to D. W. Levine et al. Additional work by D. W. Levine et al. indicated that such microcarrier treatments as that described above can be eliminated if the charge capacity of the micro- carrier is adjusted or controlled within a certain range, thereby resulting in good growth of a wide variety of anchorage-dependent cells. Such range is from about 0.1 to about 4.5 milliequivalents per gram of dry material. The microcarriers, however, still appear to be positively charged, and the most suitable microcarrier apparently is DEAE-Sephadex. U.S. Patent No. 4,189,534 to D. W. Levine et al., or British Patent Specification No. 1,535,150 to Massachusetts Instititue of Technology. See also D. W. Levine, Ph.D. Thesis, "Production of Anchorage-Dependent Mammalian Cells on Microcarriers," Massachusetts Institute of Technology, 1977. Of course, materials other than DEAE-Sephadex have been employed as microcarriers. See, for example, U.S. Patent No. 3,717,551 to B. Bizzini et al., which discloses the cultivation of cells on porous silica spherules. Moreover, the immobilization of cells for culti vation purposes has been achieved by means other than through the use of microcarriers, such as by entrapping the cells within a polymeric or gel-like matrix. 3y way of illustration only, see P. Brodelius et al., FEBS Letters, 103, 93 (1979), which describes the entrapment of plant cells within alignate beads.
Finally, the use of gelatin, collagen, and related materials in the cultivation of cells is known. For example, U.S. Patent No. 4,169,761 to P. Precausta et al. discloses the cultivation of cells on collagen fibers which are dispersed or suspended in the nutrient medium. The use of collagen in the form of gel particles for the cultivation of cells on microcarrier surfaces also has been reported; the same report mentions similar uses for cellophane, cellulose sponge, and DEAE-Sephadex, among other materials, c-b. C. Horng, Ph.D. Dissertation,
"Primary Culture of Mammalian Cells on Microcarrier Surface," Graduate School, State University of New York at Buffalo, 1975, pp. 4, 5, 136, and 138. See also, J. Leighton, "Collagen-Coated Cellulose Sponge," in P. E. Kruse, Jr., Ed., "Tissue Culture: Methods and Applica tions," Academic Press, New York, 1973, pp. 367-371 [Chem. Abstr., 81, 23770n (1974)].
According to French Patent No. 2,419,321, DEAE- Sephadex beads can be pretreated with calf fetus serum to reduce the loss of the inoculum and the lack of reproducibility in cell cultivation procedures using DEAE- Sephadex microcarriers.
It perhaps should be noted that microbial cells have been incorporated in glutaraldehyde-crosslinked gelatin to give an immobilized glucose isomerase. U.S. Patent No. 4,191,810 to N. Yoshikazu et al. Also, the fractionation and manipulation of cells have been accomplished through the use of fiber fractionation techniques - the fiber typically is nylon, optionally coated with, gelatin or a gelatin derivative which permits cell recovery by the simple expedient of melting the gelatin or gelatin derivative. See, e.g., G. M. Edelman and U. Rutishauser, "Specific Fractionation and Manipulation of Cells with Chemically Derivatized Fibers and Surfaces," in W. B. Jakoby and M. Wilchek, Editors, "Methods in
Enzymology, " Vol. 34, Academic Press, New York 1974, pp. 195-209.
With respect to harvesting or removing cells from microcarriers, the use of trypsin is well known. See, e.g., D. W. Levine et al. in "Cell Culture and Its Applications," Academic Press, New York, 1977, p. 207; U.S. Patent Nos. 4,036,693 and 3,717,551; and French Patent No. 2,419,321.
Disclosure of Invention It therefore is an object of the present invention to provide a microcarrier suitable for use in the immobilization and cultivation of anchorage-dependent animal cells and having a reduced cost.
It also is an object of the present invention to provide a microcarrier suitable for use in the immobilization and cultivation of anchorage-dependent animal cells, from which microcarrier such cells are readily removed if required. A further object of the present invention is to provide a magnetic microcarrier suitable for use in the immobilization and cultivation of anchorage-dependent animal cells, which microcarrier has a reduced cost and from which microcarrier such cells are readily removed if required.
Yet another object of the present invention is to provide a method for entrapping various animal cell types in alginate or agarose gels.
Still another object of the present invention is to provide a method for the ready removal of anchorage- dependent animal cells from the microcarriers of the present, invention.
These and other objects will be apparent to one having ordinary skill in the art from a consideration of the disclosure and claims which follow.
Accordingly, the present invention provides a method for the immobilization of animal cells, characterized in that anchorage-dependent cells are adsorbed on a micro- carrier which is enzymatically degradable without significant destruction of cell surfaces.
The present invention also provides a microcarrier for the immobilization of animal cells, characterized in that said microcarrier is enzymatically degradable without significant destruction of cell surfaces and that upon enzymatic degradation of said microcarrier having anchorage-dependent animal cells adsorbed thereon, said cells are released intact.
The present invention further provides a method for the immobilization of animal cells capable of cell division, characterized in that said cells are entrapped in a carrier comprising alginate or agarose. In a preferred embodiment, the microcarrier is a naturally occurring protein or polysaccharide or derivative thereof which is essentially water-insoluble at ambient temperature.
Brief Description of Drawings
Figure 1 illustrates the growth of two primary cell cultures on the gelatin beads of the present invention. Figure 2 illustrates the release of microcarrier- bound cells using trypsin, collagenase, and dispase, respectively.
Modes for Carrying Out the Invention
In the course of our studies to obtain a suitable gel matrix for entrapment of animal cells, we found, in analogy to T. Elsdale and J. Bard, J. Cell Biol., 54, 626-637 (1972), that collagen substrate adhered to ceils. On further investigation, a simple technique leading to the preparation of solid beads, 100-250 μm, of the closely related gelatin (the product obtained on boiling collagen) was developed. All the cells tested attached and proliferated. In this context, magnetic gelatin beads were also prepared and, as expected, no adverse effect on cell growth was observed. Potentially, such supports would permit facile recovery when used in media of viscous or particulate nature. Furthermore, it is conceivable to suspend the beads by applying an outer magnetic field, thereby minimizing the need for vigorous agitation when using carriers of high specific weight. Beads of ubiquitous chitosan, a partially deacetylated product of the polysaccharide chitin, were also prepared using a similar procedure and found to lead to attachment of cells. Their properties as supports for cell growth will be tested further. In our investigations to find supports for cell growth of anchorage- dependent cells, we coupled polylysine covalently to CNBr-activated agarose beads. Although nothing definite can be said at this point on the attachment capacity of such preparations for the cell types tested, it was found that agarose alone activated with high concentra tions of CNBr did lead to attachment and growth of cells, probably due to the positive charge introduced on the matrix.
In our experiments involving detachment of cells, we attempted an alternative approach as there was the possibility with the new microcarriers used to enzymic ally dissolve the support directly. Both dispase and collagenase dissolved the beads, leading in the case of dispase to a clear solution. In contrast to trypsin treatment which involves destruction of the cell sur face to accomplish cell removal, the approach taken here should leave the entire cells intact and viable which is an advantage, e.g., when the cells are used for immunization or when analysis of surface antigens will be done. Summarizing, as gelatin is inexpensive and repre- sents a more 'natural' surface for cell attachment than those commercially available, it appears that such microcarriers will turn out to be valuable supports for cell growth as also strongly indicated by the spinner culture experiments reported here. The ease of cell release adds to the merits of such microcarriers. Furthermore, the microcarriers can be degraded substantially completely, thereby preventing the readsorption of the cells.
The studies reported here on the entrapment of animal cells in gels were initiated because such immobilized preparations should offer the same advantages as those observed with microorganisms or plant cells. For instance, isolated cells, when immobilized in the entrapped state, would allow convenient assay in analytical metabolic perfusion studies or could be used for the enzymic conversion of certain metabolites by analogy to steroid transformation using entrapped bacteria [P.-O. Larsson et al., Nature, 263, 796-797 (1976)] or be utilized for, e.g., hormone production. These data demonstrate that cell entrapment leaves at least the major part, if not all, of their metabolic machinery intact. We applied particularly one of the more recently developed entrapping techniques, i.e., inclusion in calcium alginate [M. Kierstan and C. Bucke, Biotechnol. Bioeng., 19, 387-397 (1974); J. Klein and F. Wagner, DECHEMA-Monographien, 82, 142 (1979); and S. Ohlson et al., Eur. J. APPI. Microbiol. Biotechnol., 7, 103-110 (1979)].
It could be demonstrated with isolated rat hepatocytes that the cells remained at least partially intact on immobilization, as disruption of the cell membrane would have lead to leakage of the enzyme, lactate dehydrogenase. Subsequently, islets of Langerhans from mouse pancreas were immobilized by the same procedure, giving preparations still capable of insulin production/secretion. We also showed that adipocytes, entrapped in either calcium alginate or agarose, were still capable of metabolizing added radioactive glucose to fatty acids and could be stimulated further on addition of insulin. A drawback of the calcium alginate procedure is the requirement for Ca 2+ or similar ions to keep the network intact and, following this, the necessity to operate in phosphate-free media. Entrapment in agarose particles, as described here and applied in the studies of free fatty acid release of adipocytes, may offer an alternative approach.
With regards to anchorage-dependent cells, good microcarriers including those described here are at hand, whereas for suspension cultures entrapment appears the best alternative for immobilization. Furthermore, anchorage-dependent cells might profit from entrapment within a three-dimensional network while adsorbed to their normal carrier as this would give some protection against, e.g., shear force. It may even be that anchor age-dependent cells for which no suitable microcarrier has yet been found may accept the polymer network as recipient attachment surface or at least remain viable as demonstrated here using trypan dye exclusion tests. Although no clear cell proliferation has yet been ob served with the various entrapped cell types, they remain viable for a considerable time and permit metabolic studies, enzymic transformations or syntheses to be carried out.
The present invention is further illustrated by the example which follows. Such example, however, is not be be construed as limiting the present invention in any manner.
Example A. MATERIALS AND METHOD A.1. Materials
Chitosan, agarose (type VII), alginic acid (sodium salt, type IV), collagenase (type I, 190 U/mg) were obtained from Sigma. Glutaraldehyde (25%) was from Merck AG. Cytodex and Sepharose CL-6B were products of Pharmacia, Sweden, and the magnetic particles, Ee-04 (5 μm), were a gift from Hδganas, Sweden. Arlacεl 83 was obtained from Atlas Chemical Co., dispase (grade II, 0.5 U/mg) was from Boehringer. Gelatin (commercial grade) was from Kebo, Sweden. A.2. Cell Types
The following cells were kindly provided by Professor O. Sjδgren: the established cell lines HeLa and K562 (erythroleukemic), and the primary cell cultures S 157N (human skin fibroblasts), S 158A (human kidney carcinoma), DMH W49 (rat. colon carcinoma) and DME W1073 (rat colon carcinoma). All cells except K 562 were grown in Waymouth media supplemented with 20% fetal calf serum. X 562 were grown in SPMI medium with 10% fetal calf serum. All media were supplemented with gentamicin (50 mg/1).
A.3. Gelatin Microcarriers
A gelatin solution (10 ml; 20% (w/v)) obtained by heating to 50°C was dispersed under vigorous stirring in 100 ml of a mixture of toluene:chloroform (73/27, v/v) containing 2% (w/v) of Arlacel 83 at room temperature. After 10 min the mixture was filtered through a 100 μm nylon net whence, the collected microcarriers were transferred to acetone. After careful washing with acetone the microcarriers were evaporated to dryness. In order to obtain beads which would resist higher temperatures, cross-linking with glutaraldehyde was carried out. Dry beads (1.5 g) were reswollen in 100 ml water followed by the addition of 20 ml 25% glutaraldehyde. After gentle stirring for 30 min the beads were collected and washed with 0.15 M NaCl on a 100 μm nylon net. The beads were dispersed in 0.15 M NaCl and autoclaved at 120°C for 15 min. In order to get a suitable size distribution of the beads, they were first filtered through a 250 μm nylon net and the beads in the filtrate were then collected on a 100 μm net. They were then transferred to 0.15 M NaCl and sterilized through autoclaving at 120°C for 15 min. Magnetic microcarriers were obtained by the same procedure except that in addition to 9 ml 20% (w/v) gelatin solution, 1 g Fe3O4 particles were added prior to dispersion in the organic phase. A.4. Chitosan Microcarriers
A 2% (w/v) solution of chitosan in 1% formic acid was obtained by mixing chitosan with 1% formic acid and stirring overnight, followed by filtration of any undis solved material through a 250 μm nylon net. The chitosan solution (18 ml) was mixed with 2 ml 25% glutaraldehyde and dispersed under vigorous stirring in the same organic phase as described for gelatin microcarriers. After 15 min the mixture was transferred to methanol. The beads were then washed on a glass filter with methanol and subsequently with 0.15 M NaCl. After auto claving at 120°C for 15 min (to obtain more rigid beads), the preparation was first filtered through a 250 μm nylon net after which the beads in the filtrate were collected on a 100 μm nylon net. The beads were transferred to 0.15 M NaCl and sterilized through autoclaving at 120°C for 15 min. A.5. Test for Cell Growth Before use, all microcarriers were washed with 10 vol. medium. Gelatin and chitosan microcarriers (0.25 g wet wt) were mixed with 2.5 X 105 cells (S 157N, S 157A, DMH W49 and DMH W1073, respectively) in 5 ml media in 10 ml siliconized .test tubes. They were placed in a C02-incubator (5%) at 37°C and mixed in an end-over-end shaker. The cultures were. checked daily and the medium was replaced if necessary. The gelatin microcarriers containing Fe3O4 were tested with DMH W49. Sepharose CL-6B beads activated, with different amounts of CNBr were placed in 20 mm Petri dishes and mixed with 2.5 X 10 DMH W49 cells in 3 ml medium. The cells S 157N and DMH W1073 were allowed to grow on gelatin microcarriers (1 g beads, wet wt) suspended in 50 ml spinners. The starting cell concentrations were 35,000/ml and 22,000/ ml, respectively; 60% of the medium volume was replaced each day. For determination of the cell number, duplicate samples of 1 ml suspension were taken. After washing with phosphate-buffered saline (PBS) without
Ca 2+ and Mg2+, the beads were incubated with trypsin (0.1% in PBS with 0.02% EDTA and without Ca2+ and Mg2+) for 15 min at 37°C and the released cells counted in a
A.6. An Alternative Method for Harvesting of Cells
After 6 days the spinner culture of DMH W1073 was divided into 3 equal fractions after washing as above. They were incubated with 3 ml enzyme solution, trypsin as described above and collagenase (2 mg/ml) in PBS with
Ca 2+ but without Mg2+ and dispase (4 mg/ml) in Waymouth medium at room temperature. Samples were taken at intervals and the cell concentration determined. A.7. Alginate Immobilization
Alginate (1 part 4% (w/v)) dissolved in 25 mM Hepes, 125 mM NaCl (pH 7.4) was mixed with 1 part cell suspension in medium supplemented with twice the concentration of serum. The cell alginate suspension was extruded through a 0.8 mm nozzle into a solution of 25 mM Hepes, 50 mM CaCl2, 75 mM NaCl (pH 7.4), whereby beads with av. diam. 2 mm were formed. Immobilization was carried out at room temperature. After 5 min the beads were washed with medium and transferred to a spinner flask, which was placed in a CO2-incubator at 37°C. At intervals beads were withdrawn and dissolved in 0.1 M EDTA (pH 7.4) and the cells were counted. A.8. Agarose Immobilization Agarose (1 part 4% (w/v)) dissolved in 25 mM Hepes, 125 mM NaCl (pH 7.4) placed in a waterbath at 37°C was mixed with 1 part cell suspension in medium supplemented with twice the concentration of serum. This solution was made into beads by moulding it in a form made of Teflon. Solution was poured over a Teflon plate tightly covered with 3 mm holes. Another plate was used as support and the two were held together by clamps. Before moulding, the form was warmed to 37°C. After the agarose had solidified the form was taken apart and the 'cylindrical beads' were taken out. The beads were put in medium in a spinner flask and placed in a CO2-incubator at 37°C. At intervals beads were taken out and dissolved by heating to 70°C and the cells were counted. A.9. Preparation of Entrapped β-Cells of Islets of Langerhans
Ninety isolated islets of Langerhans (av. diam. ~ 0.1 mm) were prepared from rat [I. Lundquist, Enzyme, 12, 647-657 (1971)] pancreas and washed with Krebs- Ringer buffer lacking phosphate [I. Lundquist, supra]. The cells were suspended in 0.125 ml buffer and mixed with 0.5 ml 2.5% (w/v) sodium alginate in the same buffer. The cell alginate suspension was extruded into a solution of the buffer containing 50 mM CaCl2. After 15 min the beads were washed 3 X 10 ml with the buffer. Visual inspection showed that the beads contained an average of one islet/bead.
The scintillation vials were each filled with 10 beads and 2 ml buffer, gassed for 1 min with carbogen (95% O2, 5% CO2) and preincubated for 30 min at 37°C in a waterbath with shaking. After addition of 1 mg glucose to the vials, they were gassed and 0.1 ml samples taken after 0, 30, 60 and 120 min. The samples were frozen and stored until analyzed for insulin using radioimmunoassay. Visual inspection showed no release of the islets into the medium. A.10. Preparation of Entrapped Adipocytes
Rat adipocytes were prepared according to J. Gilemann, Diabetologia, 2 , 382-388 (1967). For entrapment in alginate the cells were transferred to 20 mM Hepes, 130 mM NaCl, 2% (w/v) albumin (pH 7.4) mixed with an equal volume of 2% (w/v) alginate (autoclaved for 15 min at 120°C) and extruded into a solution of 25 mM Hepes, 50 mM CaCl2, 75 mM NaCl (pH 7.4). After 5 min the beads were washed with Krebs-Ringer buffer con taining 24 mM Hepes, 0.55 mM glucose and 1% (w/v) albumin (storage medium).
With entrapment in agarose the cells were transferred to the above storage medium containing 2% (w/v) albumin and mixed with an equal volume of 5% (w/v) agarose. Beads were prepared as in section 2.8. After gelling the beads were transferred to storage medium. B. RESULTS B.1. Cell Attachment and Growth In a preliminary test, various cells were tested for their capacity to attach to different microcarriers (Table 1). All cells would attach to the various beads. Studies on cell growth were subsequently carried out with the most promising microcarrier, gelatin beads with the cells S 157N and DMH W1073. Upon plotting cell concentrations for the two primary cell cultures S 157N and DMH W1073 grown on gelatin beads versus culture time, it is seen that good growth occurred with no indication of cell death. Such plot is shown in Figure 1, which is based on the mean of two cell counts; curve A is for S 127N and curve B is for DME W1073.
In Figure 2, the results of three different en zymic procedures leading to cell detachment from gelatin microcarriers are given. As seen, both collagenase (curve A) and dispase (curve B) gave rise to a far higher total cell concentration than normally applied trypsin treatment. B.3. Cell Entrapment
I . Isolated cells : In a preliminary study to test whether animal cells would survive entrapment in a gel matrix, hepatocytes from rats were isolated [C. H. Floren and A; Nilsson, Biochem. J. , 168, 483-494 (1977)] and entrapped in calcium alginate. The obtained beads were subsequently incubated in perfusion buffer and water, respectively. Release of lactate dehydrogenase was monitored. About 10- times lower leakage of the enzyme was found in the sample kept in the perfusion buffer compared to the sample in water. Subsequent incubation in water of the beads previously kept in perfusion buffer, however, led to considerable leakage of active lactate dehydrogenase. Subsequently, islets of Langerhans containing β-cells were isolated and entrapped following the same procedure to check whether the latter were capable of insulin production/secretion in the immobilized state. Over 0, 30, 60 and 120 min, samples were withdrawn from the medium and assayed for insulin content using radioimmunoassay analysis. The amount of insulin secreted was 1, 23, 40 and 78 immunoreactive insulin units (μU/ml), respectively.
Finally adipocytes were isolated and entrapped in both Ca-alginate as well as agarose. They were subsequently tested for their capability to incorporate [3-3H] glucose into lipids [A. J. Moody et al., Horm. Metab. Res., 6, 12-16 (1974)] following insulin stimulation as well as for their ability to release free fatty acids by noradrenaline stimulation [N. 0. Nilsson and P. Belfrage, J. Lipid Res., 20, 557-560 (1979)]. It was found that, at the outset, the immobilized adipocyte preparation showed a higher basal incorporation than free cells which was more pronounced with cells immobilized in Ca2+-alginate. On stimulation with insulin, both preparations showed an increase in glucose incorporation (when higher concentrations of alginate were used, the cells did-not respond to insulin in the glucose test). The free fatty acid release was only studied with agarose-immobilized adipocytes as the alginate beads were too soft. The immobilized cells also showed a response to added noradrenaline by releasing free fatty acids after about twice the time required for free cells, and at ~1/3rd the rate for free cells.
II. Cell cultures: A number of cell types, i.e., fibroblasts, HeLa, DMH W49 and K 562, the latter growing in suspension, were entrapped in gels to test whether they would grow within a gel-matrix or remain viable. The gels prepared were beads of: (a) 2% alginate; (b) 2% agarose; (c) a mix ture of 2% agarose and 0.5% alginate; and (d) 2% agarose containing simultaneously entrapped Cytodex particles to which cells already had been attached. In all cases serum was entrapped simultaneously with the cells as in-diffusion of some of the components of serum was likely to be hindered. No proliferation of cells tested was observed under the conditions used; however, on average, 10-30% of the cells remained viable as judged by the trypan blue exclusion test, after 1 week of incubation. In controls run with HeLa cells, non-entrapped but kept free in solution in siliconized tubes, they had disappeared after
It is to be understood that the foregoing de tailed description is given merely by way of illustration and that many variations may be made thereon without departing from the spirit and scope of the invention.
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|Clasificación internacional||C12N11/04, B60P1/46, C12N5/00, C12N5/02, C12N11/10, C12N11/02, C12P21/02|
|Clasificación cooperativa||C12N5/0075, C12N5/0012, C12N2533/54, C12P21/02, C12N11/04, C12N2533/10, C12N2533/72, C12M25/16|
|Clasificación europea||C12N5/00S1, C12P21/02, C12N5/00C, C12N11/04|
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